The present invention relates to a substrate and a method for determining and/or monitoring electrophysiological properties of ion channels of ion channel-containing structures, typically lipid membrane-containing structures such as cells, by establishing an electrophysiological measuring configuration in which a cell membrane forms a high resistive seal around a measuring electrode, making it possible to determine and monitor a current flow through the cell membrane. The substrate is typically part of an apparatus for studying electrical events in cell membranes, such as an apparatus for carrying out patch clamp techniques utilised to study ion transfer channels in biological membranes. More particularly, the invention relates to a substrate for such patch clamp apparatus having high through-put and utilising only small amounts of test compounds, only small amounts of liquid carrier, and being capable of carrying out many tests in a short period of time by performing parallel tests on a number of cells simultaneously and independently.
The general idea of electrically insulating a patch of membrane and studying the ion channels in that patch under voltage-clamp conditions was outlined by Neher, Sakmann, and Steinback in xe2x80x9cThe Extracellular Patch Clamp, A Method For Resolving Currents Through Individual Open Channels In Biological Membranesxe2x80x9d, Pflueger Arch. 375; 219-278,1978. They found that, by pressing a pipette containing acetylcholine (ACh) against the surface of a muscle cell membrane, they could see discrete jumps in electrical current that were attributable to the opening and closing of ACh-activated ion channels. However, they were limited in their work by the fact that the resistance of the seal between the glass of the pipette and the membrane (10-50 Mxcexa9) was very small relative to the resistance of the channel (10 Gxcexa9). The electrical noise resulting from such a seal is inversely related to the resistance and was large enough to obscure the currents flowing through ion channels, the conductance of which are smaller than that of the ACh channel. It also prohibited the clamping of the voltage in the pipette to values different from that of the bath due to the large currents through the seal that would result.
It was then discovered that by fire polishing the glass pipettes and by applying suction to the interior of the pipette a seal of very high resistance (1-100 Gxcexa9) could be obtained with the surface of the cell. This Giga-seal reduced the noise by an order of magnitude to levels at which most channels of biological interest can be studied and greatly extended the voltage range over which these studies could be made. This improved seal has been termed a xe2x80x9cgiga-sealxe2x80x9d, and the pipette has been termed a xe2x80x9cpatch pipettexe2x80x9d. A more detailed description of the giga-seal may be found in O. P. Hamill, A. Marty, E. Neher, B. Sakmann and F. J. Sigworth: Improved patch-clamp techniques for high resolution current recordings from cells and cell-free membrane patches. Pflxc3xcgers Arch. 391, 85-100, 1981. For their work in developing the patch clamp technique, Neher and Sakmann were awarded the 1991 Nobel Prize in Physiology and Medicine.
Ion channels are transmembrane proteins which catalyse transport of inorganic ions across cell membranes. The ion channels participate in processes as diverse as gener-ating and timing action potentials, synaptic transmission, secretion of hormones, contraction of muscles, etc. Many drugs exert their specific effects via modulation of ion channels. Examples are antiepileptic compounds like phenytoin and lamotrigine which block voltage-dependent Na+-channels in the brain, antihypertensive drugs like nifedipine and diltiazem which block voltage dependent Ca2+-channels in smooth muscle cells, and stimulators of insulin release like glibenclamide and tolbutamide which block an ATP-regulated K+-channel in the pancreas. In addition to chemically induced modulation of ion-channel activity, the patch clamp technique has enabled scientists to perform manipulations with voltage dependent channels. These techniques include adjusting the polarity of the electrode in the patch pipette and altering the saline composition to moderate the free ion levels in the bath solution.
The patch clamp technique represents a major development in biology and medicine, since this technique allows measurement of ion flow through single ion channel proteins, and also allows the study of the single ion channel responses to drugs. Briefly, in standard patch clamp technique, a thin (app. 0.5-2 xcexcm in diameter) glass pipette is used. The tip of this patch pipette is pressed against the surface of the cell membrane. The pipette tip seals tightly to the cell and isolates a few ion channel proteins in a tiny patch of membrane. The activity of these channels can be measured individually (single channel recording) or, alternatively, the patch can be ruptured allowing measurements of the channel activity of the entire cell membrane (whole cell recording). High-conductance access to the cell interior for performing measurements can be obtained, e.g., by rupturing the membrane by applying subatmospheric pressure in the pipette.
During both single channel recording and whole-cell recording, the activity of individual channel subtypes can be characterised by imposing a xe2x80x9cvoltage clampxe2x80x9d across the membrane. In the voltage clamp technique the membrane current is recorded at a constant membrane potential. Orxe2x80x94to be more precisexe2x80x94the amplifier supplies exactly the current, which is necessary to keep the membrane potential at a level determined by the experimenter. Hence, currents resulting from opening and closing of ion channels are not allowed to recharge the membrane.
FIG. 1 shows a simplified diagram of the basic operation of a standard prior art voltage clamp amplifier such as the EPC-9 amplifier from HEKA Elektronik. An electrode 6 inside a pipette 4 is connected to the negative terminal of a feedback amplifier, while the clamping voltage (referred to a grounded bath electrode (8)) is connected to a positive terminal (from Stim. In.) and made available at a voltage monitor output. Since the measured pipette voltage and the clamp voltage are supposed to be identical, a correction potential is constantly supplied at the pipette electrode as a current forced through the large feedback resistor. After inversion, the current is made available as an analogue voltage at the Current Monitor output.
The time resolution and voltage control in such experiments are impressive, often in the msec or even xcexcsec range. However, a major obstacle of the patch clamp technique as a general method in pharmacological screening has been the limited number of compounds that could be tested per day (typically no more than 1 or 2). Also, the very slow rate of solution change that can be accomplished around cells and patches may constitute a major obstacle.
A major limitation determining the throughput of the patch clamp technique is localisation and clamping of cells and pipette, and the nature of the feeding system, which leads the dissolved compound to cells and patches.
In usual patch clamp setups, cells are placed in experimental chambers which are continuously perfused with a physiological salt solution. The establishment of the cell-pipette connection in these chambers is time-consuming and troublesome. Compounds are applied by changing the inlet to a valve connected to a small number of feeding bottles. The required volumes of the supporting liquid and the sample to be tested are high. High throughput systems for performing patch clamp measurements have been proposed, which typically consist of a substrate with a plurality of sites adapted to hold cells in a measuring configuration where the electrical properties of the cell membrane can be determined.
U.S. Pat. No. 5,187,096, Rensselaer, discloses an apparatus for monitoring cell-substrate impedance of cells. Cells are cultured directly on the electrodes which are then covered with a plurality of cells, thus, measurements on individual cells can not be performed.
WO 98/54294, Leland Stanford, discloses a substrate with wells containing electrode arrays. The substrate with wells and electrodes (metal electrodes) is made of silicon using CVD (Chemical Vapor Deposition) and etching techniques and comprises Silicon Nitride xe2x80x9cpassivationxe2x80x9d layers surrounding the electrodes. The cells are cultivated directly on the electrode array. The substrate is adapted to measure electrophysiological properties and discloses a variety of proposed measuring schemes.
WO 99/66329, Cenes, discloses a substrate with perforations arranged in wells and electrodes provided on each side of the substrate. The substrate is made by perforating a silicon substrate with a laser and may be coated with anti-adhesive material on the surface. The substrate is adapted to establish giga seals with cells by positioning the cells on the perforations using suction creating a liquid flow through the perforations, providing the anti-adhesion layer surrounding the perforations, or by guiding the cells electrically. The cells can be permeabilised by EM fields or chemical methods in order to provide a whole-cell measuring configuration. All perforations, and hence all measurable cells, in a well share one working electrode and one reference electrode, see FIG. 1, hence measurements on individual cells can not be performed.
WO 99/31503, Vogel et al., discloses a measuring device with an aperture arranged in a well on a substrate (carrier) and separating two compartments. The measuring device comprises two electrodes positioned on either side of the aperture and adapted to position a cell at the aperture opening. The substrate may have hydrophobic and hydrophilic regions in order to guide the positioning of the cells at the aperture opening.
The present invention provides a substrate and a method optimised for determining or monitoring current flow through ion channel-containing structures such as cell membranes, with a high throughput and reliability and under conditions that are realistic with respect to the influences to which the cells or cell membranes are subjected. Thus, the results determined using the substrate and the method of the invention, e.g., variations in ion channel activity as a result of influencing the cell membrane with, e.g., various test compounds, can be relied upon as true manifestations of the influences proper and not of artefacts introduced by the measuring system, and can be used as a valid basis for studying electrophysiological phenomena related to the conductivity or capacitance of cell membranes under given conditions.
This is because the current through one or more ion channels is directly measured using reversible electrodes as characterized below, typically silver/silver halide electrodes such as silver chloride electrodes, as both measuring electrodes and reference electrodes.
The substrate and method of the invention may be used not only for measurements on cell membranes, but also on other ion channel-containing structures, such as artificial membranes. The invention permits performing several tests, such as electrophysilogical measurements on ion transfer channels and membranes, simultaneously and independently. The substrate of the invention constitutes a complete and easily handled microsystem which uses only small amounts of supporting liquid (a physiological salt solution, isotonic with the cells, that is, normally having an osmolarity of 150 millimolar NaCI or another suitable salt) and small amounts of test samples.
In one aspect, the invention relates to a plane substrate having an first surface part and an opposite second surface part, the first surface part having a plurality of sites each of which is adapted to hold an ion channel-containing structure, each site having a measuring electrode associated therewith, the substrate carrying one or more reference electrodes, the measuring electrodes and the respective reference electrode or reference electrodes being electrodes capable of generating, when in electrolytic contact with each other and when a potential difference is applied between them, a current between them by delivery of ions by one electrode and receipt of ions by the other electrode, each of the sites being adapted to provide a high electrical resistance seal between an ion channel-containing structure held at the site and a surface part of the site, the seal, when provided, separating a domain defined on one side of the ion channel-containing structure and in electrolytic contact with the measuring electrode from a domain defined on the other side of the ion channel-containing structure and in electrolytic contact with the respective reference electrode so that a current flowing through ion channels of the ion channel-containing structure between the electrodes can be determined and/or monitored, the electrodes being integrated with the substrate and having been formed by a wafer processing technology.
In another aspect, the invention relates to a method method of establishing a whole cell measuring configuration for determining and/or monitoring an electrophysiological property of one or more ion channels of one or more ion channel-containing structures, said method comprising the steps of
providing a substrate as defined above,
supplying a carrier liquid at one or more sites, said carrier liquid containing one or more ion channel-containing structures,
positioning at least one of the ion channel-containing structures at a corresponding number of sites,
checking for a high electrical resistance seal between an ion channel-containing structure held at a site and the surface part of the site with which the high electrical resistance seal is to be provided by successively applying a first electric potential difference between the measuring electrode associated with the site and a reference electrode, monitoring a first current flowing between said measuring electrode and said reference electrode, and comparing said first current to a predetermined threshold current and, if the first current is at most the predetermined threshold current, then approving the site as having an acceptable seal between the ion cannel-containing structure and the surface part of the site, and
establishing a whole-cell configuration at approved sites,
whereby a third current flowing through ion channels of the ion channel-containing structure between the measuring electrode and the reference electrodes can be determined and/or monitored.
An ion channel-containing structure in a solution may be guided towards a site on a substrate either by active or passive means. When the ion channel-containing structure makes contact with the site, e.g. substrate around an electrode, the contact surfaces form a high electrical resistance seal (a giga-seal) at the site, e.g. surrounding the electrode, so that an electrophysiological property of the ion channels can be measured using the respective electrode. Such electrophysiological property may be current conducted through the part of membrane of the ion channel-containing structure that is encircled by the giga-seal.
In the present context, the term xe2x80x9cgiga-sealxe2x80x9d normally indicates a seal of a least 1G ohm, and this is the size of seal normally aimed at as a minimum, but for certain types of measurements where the currents are large, lower values may be sufficient as threshold values.
The whole-cell configuration may be obtained by applying, between the measuring electrode associated with each approved site and a reference electrode, a series of second electric potential difference pulses, monitoring a second current flowing between the measuring electrode and the reference electrode, and interrupting the series of second electric potential difference pulses whenever said second current exceeds a predetermined threshold value, thereby rupturing the part of the ion channel-containing structure which is closest to the measuring electrode.
Alternativelly, the whole-cell configuration may be obtained by subjecting the part of the ion channel-containing structure which is closest to the measuring electrode to interaction with a pore forming substance.
It should be noted that in the present context, the term xe2x80x9cwhole-cell configurationxe2x80x9d denotes not only configurations in which a whole cell has been brought in contact with the substrate at a measuring site and has been punctured or, by means of a pore-forming substance, has been opened to electrical contact with the cell interior, but also configurations in which an excised cell membrane patch has been arranged so that the outer face of the membrane faces xe2x80x9cupwardlyxe2x80x9d, towards a test sample to be applied.
As the measuring electrode associated with a site is one of a plurality of electrodes on the substrate, and the ion channel-containing structure is one of many in a solution, it is possible to obtain many such prepared measuring set-ups on a substrate. A typical measurement comprises adding a specific test sample to the set-up, for which reason each measuring set-up is separated from other measuring set-ups to avoid mixing of test samples and electrical conduction in between set-ups.